DNA sequence analysis of hprt mutants persisting in peripheral blood of cynomolgus monkeys more than two years after ENU treatment

Environ Mol Mutagen. 1999;33(1):42-8.


We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T-lymphocytes. This primate model allows us to study mutations and their kinetics under well-controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg). At 832 days after that first treatment, the monkey received a second dose of 77 mg/kg ENU. Up to 1,331 days after the second treatment, the T-cell mutant frequency (44.2 x 10(-6)) was still 26-fold higher than background (1.7 x 10(-6)), suggesting that mutants persisted in the peripheral blood. Mutant clones from Days 974, 1,164, and 1,311 after the second treatment were selected in thioguanine. Hprt cDNA was prepared from a cell lysate, PCR-amplified, and sequenced. Of 45 mutants, 30 yielded PCR product and 26 were sequenced. Base substitutions were found in 21 (81%) of the 26 mutants and consisted of one G:C --> A:T and five A:T --> G:C transitions, one G:C --> C:G, eight A:T --> T:A, and six A:T --> C:G transversions. Therefore, most base substitutions occurred at A:T basepairs, characteristic of ENU-induced mutations in vivo, and were detected up to 3.6 years after the second treatment. Deletions of exons 2 and 3 occurred in two mutants and exon 7 was deleted in one mutant. There were two insertion mutants: one was a single base insertion and the other contained an insertion of 277 basepairs which was nearly identical to a simian retroviral sequence.

MeSH terms

  • Animals
  • Clone Cells
  • Codon / genetics
  • DNA Mutational Analysis
  • Ethylnitrosourea / pharmacology*
  • Female
  • Hypoxanthine Phosphoribosyltransferase / blood*
  • Hypoxanthine Phosphoribosyltransferase / genetics*
  • Macaca fascicularis
  • Mutagenesis, Site-Directed
  • Mutagens / pharmacology*
  • Mutation*
  • Sequence Deletion
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / metabolism
  • Time Factors


  • Codon
  • Mutagens
  • Hypoxanthine Phosphoribosyltransferase
  • Ethylnitrosourea